Human erythropoietin (hEPO) is a glycoprotein which boosts production of erythrocytes by acting on erythroid progenitors to promote their differentiation into erythrocytes in human. For this reason, hEPO has been used as a medicament for the treatment of human renal anemia, as well as in preserving autologous blood in preparation for an operation. Erythropoietin is a glycoprotein consisting of 165 amino acid residues, in which the asparagine residues at positions 24, 38, and 83 from its N-terminus are modified with N-linked sugar chains, and the serine residue at position 126 with an O-linked sugar chain, respectively. Thus, the peptide chain of hEPO is modified with sugar chains at four positions. These sugar chains contribute to the stability of hEPO in the body, and hEPO stripped of those sugar chains shows little activity because it is rapidly decomposed in the body.
Aiming to increase the stability of hEPO in the body, various attempts have been made to build mutant-type hEPOs having altered amino acid sequence so as to be modified by an increased number of sugar chains. Darbepoetin is one of such mutant-type hEPOs. Darbepoetin is a mutant-type hEPO which has mutations introduced at five positions in its peptide chain consisting of 165 amino acid residues: Ala30Asn, His32Thr, Pro87Val, Trp88Asn, and Pro90Thr (Patent Document 1). Among these mutations, asparagine residues at two positions, Ala30Asn and Trp88Asn, provide fresh sites for binding of N-linked sugar chains. Since wild-type hEPO possesses four sites for binding of sugar chains as mentioned above, darbepoetin consequently has six sites in total for binding of sugar chains. Actually, darbepoetin produced using CHO cells, which originate from Chinese hamster ovary, is modified at those six positions with sugar chains. Such a form of darbepoetin was approved as an ethical drug in the name of Nesp (registered trademark) in 2010 in Japan.
The generally recommended dosage of Nesp is weekly 20 μg i.v. as the starting dose, then biweekly 30-120 μg i.v. as the maintenance dose for a patient receiving hemodialysis; and for a patient receiving peritoneal dialysis or a predialysis patient with chronical renal failure, biweekly 30 μg s.c. or i.v. as the starting dose, then 60-180 μg s.c. or i.v. every four weeks as the maintenance dose. Compared with other erythropoietin preparations which generally require administration two or three times a week, Nesp lessens patient's burden by reducing administration frequency.
As a method for production of mutant type erythropoietin using CHO cells, a method is reported, in which a mutant erythropoietin expressed by culturing transformed CHO cells in a serum-free medium is purified by a four-step chromatography using a mixed mode column (Capto Adhere column), a first cation exchange column, a second cation exchange column, and anion exchange column (or Capto Adhere column)(Patent Documents 2-3). The method in this report is aimed to isolate an isoform of mutant erythropoietin having a low isoelectric point, and characterized in that the mutant erythropoietin is first adsorbed by the resin in the mixed mode column process and the resin then is washed in an acidic condition (pH 4.0).
Further, as for a method for production of a mutant type erythropoietin using transformed CHO cells, there is reported a method, in which the mutant erythropoietin expressed by culturing such CHO cells is purified by a three-step chromatography using an affinity column based on lectin/m-aminophenyl matrix, an anion exchange column, and a hydrophobic column (Patent Document 4). Furthermore, a method for purification of a mutant-type erythropoietin expressed by culturing transformed CHO cells in a serum-free medium is reported, which method employs a hydrophobic column, an anion exchange column, a weak cation exchange column, and a strong cation exchange column (Patent Document 5). Furthermore, a method for purification is reported in which a mutant-type erythropoietin expressed by transformed CHO cells in a serum-free medium is purified through a three-step chromatography employing a first anion exchange column, a hydroxyapatite column, and a second anion exchange column (Patent Document 6). Still further, there are reported a method in which a mutant-type erythropoietin expressed by culturing transformed CHO cells is purified through a three-step chromatography using a dye-affinity column, a hydroxyapatite column, and an anion exchange column (Patent Document 7), and a method for purification of mutant-type human erythropoietin which includes a step of washing an anion exchange column in which the mutant-type erythropoietin is adsorbed with an arginine-containing buffer (Patent Document 8).